Anti-inflammatory actions of neuroprotectin d1/protectin d1 and it&#39;s natural stereoisomers

ABSTRACT

(Neuro)protectin D1 (1OR,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) and 15,16-dehydro-PD1 and their derivatives are useful in the treatment of airway inflammation, especially asthma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) to U.S. Ser.Nos. 60/723,052, filed Oct. 3, 2005 (Attorney docket number 187127/US),entitled “Anti-Inflammatory Actions of Neuroptectin D1/Protectin D1 andIts Natural Stereoisomers” and 60/749,786, filed Dec. 13, 2005 (Attorneydocket number 187127/US/2), entitled “Anti-Inflammatory Actions ofNeuroptectin D1/Protectin D1 and Its Natural Stereoisomers”, thecontents of both are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work leading to this invention was supported in part by NationalInstitutes of Health (NIH) grants GM38765, P50-DE016191, HL068669 andAI068084. The U.S. Government therefore may have certain rights in theinvention.

FIELD OF THE INVENTION

Protectin D1, neuroprotectin D1 when generated by neural cells, is amember of a new family of bioactive products generated fromdocosahexaenoic acid (1-3). The complete stereochemistry of protectin D1(10,17S-docosatriene), namely chirality of the carbon 10 alcohol andgeometry of the conjugated triene, required for bioactivity remained tobe assigned. To this end, PD1 generated by human neutrophils duringmurine peritonitis and neural tissues was separated from natural isomersand subject to LC-MS-MS and GC-MS. Comparisons with six10,17-dihydroxydocosatrienes prepared by total organic and biogenicsynthesis showed that PD1 from human cells carrying potent bioactivityis 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid.Additional isomers identified included trace amounts of Δ15-trans-PD1(isomer III), 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoicacid (isomer IV), and a double dioxygenation product10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomerI), present in exudates. ¹⁸O₂ labeling showed that 10S,17S-diHDHA(isomer I) carried ¹⁸O in the 10-position alcohol, indicating sequentiallipoxygenation, whereas PD1 formation proceeded via an epoxide. PD1 at10 nM attenuated (˜50%) human neutrophil transmigration whileΔ15-trans-PD1 was essentially inactive. PD1 was a potent regulator ofPMN infiltration (˜40% at 1 ng/mouse) in peritonitis. The rank order at1-10 ng dose was PD1≈PD1 methyl ester >>Δ15-trans PD1>10S,17S-diHDHA(isomer I). 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoicacid (isomer VI) proved ≧PD1 in blocking PMN infiltration but was not amajor product of leukocytes. PD1 also reduced PMN infiltration afterinitiation (2 h) of inflammation and was additive with resolvin E1.These results indicate that PD1 is a potent stereoselectiveanti-inflammatory molecule.

BACKGROUND OF THE INVENTION

The resolution of inflammation is a central component of host defenseand the return of tissue to homeostasis (4). It is recognized thatinflammation plays a key role in many prevalent human diseases includingcardiovascular diseases, atherosclerosis, Alzheimer's disease, andcancer (5-7). Although much is known about the molecular basis ofinitiating signals and proinflammatory chemical mediators ininflammation, it has only recently become apparent that endogenous stopsignals are critical at early checkpoints within the temporal events ofinflammation (8). In this context, lipid mediators are of interest. Thearachidonic acid-derived prostaglandins and leukotrienes are potentpro-inflammatory mediators (9), whereas their cousins, the lipoxins,biosynthesized from arachidonic acid, are potent anti-inflammatory andproresolving molecules (for reviews see 10, 11, 12). During the courseof inflammation, arachidonate-derived eicosanoids switch fromprostaglandins and leukotrienes within inflammatory exudates to lipoxinsthat in turn stop the recruitment of neutrophils to the site. Thisswitch in eicosanoid profiles and biosynthesis is driven, in part, bycyclooxygenase-derived prostaglandin E₂ and prostaglandin D₂, whichinstruct the transcriptional regulation of enzymes involved in lipoxinbiosynthesis (13). Hence, the appearance of lipoxins within inflammatoryexudates is concomitant with spontaneous resolution of inflammation(13), and these chemical mediators are non-phlogistic stimulators ofmonocyte recruitment and macrophage phagocytosis of apoptotic PMN (14,15)

Further studies on the endogenous mechanisms of anti-inflammation usinga murine model of spontaneous resolution demonstrated, for the firsttime, that resolution is an active biochemical process that involves thegeneration of specific new families of lipid mediators (for recentreviews, see refs. 16, 17). During spontaneous resolution, cell-cellinteractions and transcellular biosynthesis lead to the production ofthese new families of potent bioactive lipid mediators from ω-3essential fatty acid precursors and were termed resolvins (resolutionphase interaction products derived from DHA and EPA) and protectins(docosatrienes derived from DHA) ((1, 3, 18) and recently reviewed in(19)). These novel di- and trihydroxy-containing products from EPA andDHA that are generated by previously unrecognized enzymatic pathwaysdisplay potent anti-inflammatory and immunoregulatory actions in vitroand in vivo in murine models of acute inflammatory actions (1, 3, 18).

In 1929, the omega-3 polyunsaturated fatty acids were assigned essentialroles because their exclusion from the diet gave rise to a new form ofdeficiency disease (20). Many recent reports document the importance offish oil (omega-3) fatty acids EPA and DHA in human diseases associatedwith inflammation. In particular, omega-3 DHA and EPA are protective ininflammatory bowel disease and colitis (21), cardiovascular disease(22-25), and Alzheimer's disease (26). However, the molecular mechanismsresponsible for these documented beneficial actions of omega-3 fattyacids remain an important challenge. DHA is enriched in neural tissues,where it appears to play functional as well as structural roles (27,28). Along these lines, results from earlier studies indicated that DHAwas enzymatically converted to products coined docosanoids that might belinked to retinal protection (29) and neuronal function (30). Thestructures of the molecules involved, however, were not established.

Human whole blood isolated leukocytes, and glial cells enzymaticallyconvert DHA to 17S-hydroxy-containing docosatrienes and 17S-seriesresolvins (1, 3). The novel 10,17S-docosatriene, first identified inref. (3) and its basic structure established, displayed potentanti-inflammatory actions, i.e., reducing PMN numbers in exudates invivo, and down regulating production of proinflammatory cytokines byglial cells in vitro (1). During the resolution phase of peritonitis,unesterified DHA levels increase within exudates and 10,17S-docosatrieneis generated within the resolving exudates, where it appears to promotecatabasis, or the return to homeostasis, by shortening the resolutioninterval (31). Of special interest, this DHA-derived 10,17S-docosatrieneis generated in vivo during strokes in murine tissues and limits theentry of leukocytes into the area of neural damage, reducing themagnitude of tissue injury (32). It was found that 10,17S-docosatrieneis neuroprotective in retinal pigmented cells and introduced the termneuroprotectin D1 for this potent compound (2), which accumulates in theipsilateral hemisphere of the brain following focal ischemia (33).

Recent results indicate that neuroprotectin D1 is formed from DHA incornea in a lipoxygenase-dependent fashion to protect from thermalinjury as well as promote wound healing (34). It is noteworthy thatneuroprotectin D1, resolvin D1, and resolvin D5 are all produced bytrout brain cells from endogenous DHA, suggesting that the structures ofthese DHA-derived mediators are conserved from fish to humans (35).Together, these recent findings underscore the need to establish thecomplete stereochemistry of endogenous biologically active10,17S-docosatriene, namely its carbon 10 position alcohol chirality anddouble bond geometry of its conjugated triene system. In recognition ofits wide scope of formation and actions, protectin D1 (PD1) is used todenote the structure of this chemical mediator and the prefix neurobefore protectin D1 is used to note its tissue origin and address. Here,the complete stereochemistry of protectin D1 and its related naturalisomers (i.e., Δ15-trans-PD1) as well as their anti-inflammatoryproperties are reported.

Therefore, a need exists for additional understanding of how otherpolyunsaturated compounds and biological derivative may provide insightinto such complex biological pathways.

BRIEF SUMMARY OF THE INVENTION

The present invention surprisingly provides methods to isolate,substantially purify (purify) and prepare compounds such as: I10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid; II10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic-acid; V10S,17R,-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic-acid; III10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15E,19Z-hexaenoic-acid; IV10R,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic-acid; and VI10S,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic-acid. Also,derivatives such as esters, e.g., methyl esters, of the acids can beprepared and show biological activity as discussed herein.

Additionally, the 15,16-dehydro-PD1 provides a chemically stable systemthat also has biological activity.

In other aspects, the invention provides10,17-dihydroxy-docasa-hexaenoic acids having the general formula (VII):

wherein R is a hydrogen atom, an alkyl group, or is a pharmaceuticallyacceptable salt and each of P₁ and P₂, individually, is a hydrogen atomor a protecting group. The dashed line represents that the double bondcan be “cis” or “trans” in configuration. In certain aspects, compoundsI, II, IV, V and VI, each independently of each other, are excluded fromthe invention.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed descriptions are to be regarded asillustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Panel A: LC-MS-MS profiles and chromatographic behavior ofrelated isomers. Left panels: LC chromatograms plotted at UV absorbance270 nm; right panels: corresponding MS profiles obtained for m/z MS-MS359. Upper inserts: representative profiles from human PMN. Right:plotted at MS² m/z=359; another incubation plotted at m/z 261 of the MS²359. The position of synthetic III is shown for comparison. Middle:Murine exudate profiles and lower: profiles obtained for a mixture ofrelated synthetic isomers I-VI (see FIG. 2 for structures). Note thatisomers I and VI and II and V coelute in this HPLC system (also see FIG.8 and text). Panel B: MS-MS Spectrum of PD1 obtained from murineperitonitis. The LC retention time was 32.9 min for the recordedspectrum. Panel C: MS-MS spectrum of synthetic PD1. Panel D: GC-MSspectrum of derivatized PD1. MS obtained following treatment withdiazomethane and trimethylsilane. See FIG. 8 and methods section for NMRdata and conditions of analysis using LC-MS-MS and GC-MS.

FIG. 2: PD1 and related 10,17-diHDHA isomers. List of compounds preparedand used for the present experiments. PD1 obtained from biologicaltissues and incubations was identified earlier (1) as a potent bioactiveproduct generated from DHA possessing the 10,17S-dihydroxy-docosatrienestructure with a conjugated triene unit between carbons 10 and 17. Asdenoted, the configuration of the carbon 10 alcohol and double bonds ofthe conjugation remained to be determined; see text for details andMethods for the NMR values obtained for each of the isomers prepared bytotal organic synthesis.

FIG. 3: Strategy for total synthesis of PD1 and related isomers. The C₁₀and C₁₇ stereochemistry of 1 was derived from enantiomerically pureglycidol derivatives B and H which were reacted with alkynylnucleophiles derived from A and I, respectively. The (Z) alkene geometryat positions 4-5, 7-8, 15-16 and 19-20 was obtained from selectivehydrogenation of acetylenic precursors, which were constructed usingcoupling procedures. The (E) geometry at positions 11-12 and 13-14 wassecured during the synthesis of intermediate F. Other stereoisomers of 1were synthesized similarly.

FIG. 4: PD1 and related double dioxygenation products. Panel A: MS-MS ofthe 10S,17S-diHDHA (isomer I, FIG. 2) carrying ¹⁸O obtained fromincubations enriched in ¹⁸O₂ atmosphere. The substrate was 17S—H(p)DHA;hence, the 17 position alcohol retained the ¹⁶O and remained unlabeledwhile both the carbon 7 and 10 position alcohols were labeled from ¹⁸O₂.Fragments carrying ¹⁸O₂ were increased in m/z+2. Panel B: Scheme for PD1enzymatic formation: epoxidation versus dioxygenation for production ofits natural isomer. See text for details.

FIG. 5: PD1 blocks human PMN transmigration across endothelial cells.PD1, but not its Δ15-trans-PD1 isomer, inhibited LTB₄-induced PMNmigration across microvascular endothelial monolayers. Neutrophils (10⁶per monolayer) were exposed to vehicle containing buffer or at theindicated concentrations of compound for 15 minutes (see Methods).Neutrophils were then layered on HMEC monolayers and stimulated totransmigrate by addition of 10⁻⁸ M LTB₄ for 90 min at 37° C.Transmigration was assessed by quantitation of the PMN markermyeloperoxidase. PD1 represents the mean±SEM percent migration ofneutrophils compared to vehicle-treated neutrophils for 9 separatedonors and experiments, each performed in triplicate. The results withΔ15-trans PD1 isomer are the mean±SEM obtained for 6 separate PMN donorswhere each point was also in triplicate. * denotes p<0.01.

FIG. 6: Dose-dependent inhibition of acute inflammation in vivo withsynthetic PD1 and its isomers. Peritonitis was initiated in 6-8-week-oldmale FVB mice (Charles River Laboratories) by peritoneal injection of 1mg of zymosan A. Mice were injected with (Panel A): the doubledioxygenation product 10S,17S-diHDHA (Compound I, FIG. 2), synthetic PD1(Compound II), Δ15-trans-PD1 (Compound III), the 10S,17R-diHDHA isomer(Compound V), Compound VI, or vehicle alone. *p<0.05; n≧3 for eachcompound. n≧7 for PD1 and n≧4 for Compound I. Panel B: PD1, PD1 methylester, or the isomer (Compound VI) methyl ester. Peritoneal lavages wereobtained at 2 h and leukocytes enumerated. Results are expressed aspercent inhibition compared to mice injected with zymosan A (1 mg) andvehicle alone. * p<0.05; n≧4 for each compound.

FIG. 7: PD1 actions in vivo. A) PD1 treatment during the course of acuteinflammation reduces PMN infiltration. Peritonitis was induced in6-8-week-old male FVB mice (Charles River Laboratories) by peritonealinjection of 1 mg of zymosan A (♦) as in FIG. 6. Synthetic compound PD1(▴; cf. FIG. 1) free acid or its synthetic carboxy methyl ester (▪) eachat 1 ng dose/mouse were injected by peritoneal injection i.p. 2 h afterzymosan A-initiated peritonitis. 4 h after induction of peritonitis,rapid peritoneal lavages were collected, and cell-type enumeration wasperformed. * p<0.05, δ p<0.05, from zymosan plus vehicle alone. B) PD1and RvE1 have additive anti-inflammatory actions in vivo. Mice wereinjected i.p. with 10 ng/mouse of either PD1, RvE1, or both, andexudates were collected at 2 h. *p<0.05.

FIG. 8: provides physical attributes of several of the novel compounds.

FIG. 9: provides leukocyte infiltration in Murine Peritonitis.

FIG. 10: provides characteristics of test subjects.

FIG. 11: Generation of Protectin D1 in asthma. Exhaled breathcondensates were obtained from volunteer subjects in the emergencydepartment during a clinical exacerbation of asthma. Lipids wereextracted and subjected to analysis by LC-PDA-MS-MS. (a) LC chromatogramplotted for ms/ms at m/z 343 and (b) corresponding MS profile werediagnostic for 17(S)-hydroxy-DHA (i.e.,17S-hydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid). Material wasalso present in the lipid extracts with (c) LC chromatogram for m/z 217of ms/ms at m/z 359, (d) UV absorbance spectrum (inset, left) and massspectrum diagnostic for authentic Protectin D1 (i.e.,10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid). Insets,the fragmentation ions are denoted for (b) 17(S)-hydroxy-DHA and (d)Protectin D1. Results are representative of n=3.

FIG. 12: Lung histopathology from mice given PD1. Mice were sensitizedand aerosol challenged with OVA in the presence of PD1 ((a), 200 ng,(b), 20 ng, (c), 2 ng) or (d), vehicle. Representative (n≧3) lung tissuesections (magnifications: ×20 (left column), ×40 (right column)) wereobtained from fixed, paraffin-embedded lung tissue, prepared and stainedwith hematoxylin and eosin. Arrows denote representative EOS; Br,bronchus; v, vessel.

FIG. 13: PD1 decreases airway mucus. Representative lung tissue sectionsfrom mice given PD1 (a, 200 ng, b, 20 ng) or (c) vehicle were stainedwith periodic acid Schiff (magnifications: ×20 (left column), ×40 (rightcolumn). Arrows indicate representative mucus (magenta) containinggoblet cells.

FIG. 14: PD1 sharply reduces leukocyte infiltration. (a), Tissuemorphometric analyses were performed to determine the impact of PD1 onEOS accumulation in pulmonary vessels (V-EOS), large airways (Aw-EOS)and alveoli (Alv-EOS). (b), BALFs were obtained from OVA sensitized andchallenged mice. Leukocytes in BALF were enumerated and identified afterWright-Giemsa stain. Results are expressed as mean±SEM (n≧3). *P<0.05 byStudent's t-test compared to control animals.

FIG. 15: PD1 selectively decreases airway inflammatory mediators. In thepresence or absence of PD1, the mediator profile in BALF was determinedin materials from OVA sensitized and challenged mice for specific (a)cytokines (IL-13, IL-5, IL-12), and (b) lipid mediators (CysLTs, LXA₄and PGD₂). Results are expressed as mean±SEM (n≧5, d=2). *P<0.05 byStudent's t-test compared to control animals.

FIG. 16: PD1 reduces airway hyper-responsiveness. (a), OVA sensitizedmice were treated with PD1 20 ng (□), 200 ng (∘) or vehicle (▴) prior toOVA aerosol challenge. Airway reactivity was determined bymethacholine-dependent change in peak lung resistance. Results areexpressed as mean±SEM (n≧5). *P<0.05 by one-way ANOVA compared tocontrol animals. (b), ED₂₀₀ was determined for methacholine-dependentchanges in mean lung resistance for OVA-sensitized animals receiving PD1(0, 2, 20 or 200 ng) prior to OVA aerosol challenge and for controlanimals receiving buffer (PBS) instead of OVA during sensitization andchallenge phases of the model. *P<0.05 by Student's t-test compared tocontrol animals.

FIG. 17: PD1 treatment promotes resolution of allergen-driven leukocytesin mouse lung. BALFs were obtained from OVA sensitized and challengedmice that received either PD1 (20 ng, hatched bars) or vehicle (0.9%saline, black bars) for three consecutive days prior to study.Leukocytes in BALF were enumerated and identified after Wright-Giemsastain. Results are expressed as mean±SEM (n≧3). *P<0.05 by Student'st-test compared to control animals.

DETAILED DESCRIPTION

Abbreviations used are:

ω-3 PUFA, omega-3 polyunsaturated fatty acid;

5S,15S-diHETE, 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetranoic acid;

7S,17S-diHDHA, 7S,17S-dihydroxy-docosa-4Z,8E,10Z,13Z,15E,19Z-hexaenoicacid (resolvin D5);

10S—HDHA, 10S-hydroxy-docosa-hexaenoic acid;

10S,17S-docosatriene,10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (thedioxygenation product);

10,17-docosatriene isomers,10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid;10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15E,19Z-hexaenoic acid;10R,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid;10S,17R-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid;10S,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid;

17S—HDHA, 17S-hydroxy-docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid;7S-H(p)DHA, 17S-hydroxy(peroxy)-docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoicacid;

BAL, bronchoalveolar lavage;

COX-2, cyclooxygenase 2;

CysLT, cysteinyl leukotriene;

DHA, C22:6, docosahexaenoic acid;

EBC, exhaled breath condensate;

EOS, eosinophil;

GC-MS, gas chromatography mass spectrometry;

HMEC, human micro-vascular endothelial cells;

LC-UV-MS-MS, liquid chromatography-ultraviolet-tandem mass spectrometry;

LO, lipoxygenase;

LT, leukotriene;

LX, lipoxins;

Lymph, lymphocyte;

MS, mass spectrometry;

PD1, protectin D1/neuroprotectinD1,10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (whengenerated in neural tissues the prefix neuro is added, henceneuroprotectin D1 or NPD1 as in ref. 2);

PMN, polymorphonuclear leukocytes;

RP-HPLC, reverse-phase high performance liquid chromatography; and

RvE1, resolvinE1,5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid.

Neuroprotectin D1/protectin D1 (10,17-docosatriene) is a potentbioactive lipid mediator derived from DHA that displaysanti-inflammatory actions (1, 3) and is generated during the resolutionphase of an acute inflammatory response (31). The basic structure ofthis novel potent DHA-derived mediator was determined, i.e.,10,17-dihydroxydocosatriene (1, 3, 37); its potent role in neuralprotection was recently uncovered (2) and thus it is denoted asneuroprotectin D1 (NPD1) when produced in neural tissues. Given theimportance of establishing the molecular basis of endogenousanti-inflammation and natural resolution (17), as knowledge of thesepathways and mechanisms in vivo may provide new therapeutic approachesto human disease, evidence was sought for the complete stereochemistryof PD1. On the basis of physical, biosynthetic, and biologicalproperties in matching results with human cells and synthetic materials,the complete stereochemistry of PD1 was assigned10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (CompoundII; FIGS. 1, 2, and 6A and FIG. 8).

On identification of 10,17-diHDHA in resolving inflammatory exudates (3,37) and potent anti-inflamatory actions, it was critical to establishits biosynthesis from DHA. To address this, isolated human PMN, wholeblood, microglial cells, and murine exudates and tissues (1, 3) werestudied. The isolation and identification of alcohol trapping productsindicated the involvement of an epoxide intermediate in the conversionof DHA to 10,17S-diHDHA, a docosatriene containing a characteristicconjugated triene structure involving three of the six double bondspresent in this compound. The role of a 16(17)epoxide intermediategenerated from the 17S-hydroperoxy-DHA precursor was further supportedby the identification of two vicinal diols, i.e.,16,17S-dihydroxydocosatrienes present in these LC-MS-MS profiles alsogenerated from DHA (1). The 16(17)epoxy-DHA intermediate could open vianon-enzymatic hydrolysis to a racemic mixture, i.e., 16R/S,17S-diHDHA,or to a single 16,17S-vicinol alcohol by the actions of an appropriateepoxide hydrolase in a reaction similar to that demonstrated earlier inthe biosynthesis of LXA₄ (39, 44, 46). The biosynthesis of PD1 by humancells (Compound II) with this stereochemistry from a 16(17)epoxideintermediate would require an enzymatic reaction to move the double bondconfiguration to set the triene geometry to 11E,13E,15Z and direct theattack of H₂O and insertion of its oxygen into the carbon 10 position ofPD1 determined in the present experiments to be in the 10Rstereochemical configuration.

In addition to PD1 (Compound II) in human cell extracts, which carriedpotent bioactions, an isomer 10S,17S-diHDHA (Compound I) was alsoidentified in murine exudates with lesser amounts in isolated humancells (FIG. 1). Compound I was found to be a double dioxygenationproduct and was also formed from DHA but in a reaction that required twosequential lipoxygenation steps and oxygen incorporation that wasdirected at the 10 position derived from molecular oxygen (i.e., ¹⁸O₂ inan enriched atmosphere in vitro). This reaction producing 10S,17S-diHDHAis markedly different from the proposed enzymatic hydrolysis of theepoxide intermediate in mammalian tissues to produce PD1. The doubledioxygenation product formed in vivo is different from PD1 in three keyways: i) PD1 carbon 10 position alcohol is predominantly in the 10Rconfiguration while the dioxygenation product is mainly in the 10Sconfiguration; ii) the double bond structure of PD1 conjugated triene isin the 11E,13E,15Z configuration; the 10S,17S-dioxygenation productconjugated triene system is in the 11E,13Z,15E configuration; iii) mostimportantly, PD1 is more potent than the dioxygenation product; PD1(Compound II)>>10S,17S-diHDHA (Compound I); and iv) PD1 is generated byisolated human leukocytes and tissues.

Also in support of the stereospecific basis of these DHA-derivedproducts in human and murine systems is the bioaction of theΔ15-trans-PD1 isomer (Compound III), which can arise via workup-inducedisomerization of PD1 and possesses little bioactivity in vitro or invivo within the dose or concentration range (FIG. 5) observed withbiogenic or synthetic PD1 (FIGS. 6 and 7). Also, Compound IV, identifiedin human leukocytes (FIG. 1) and which differed from Compound I at the10R position and carried the same double bond geometry, was essentiallyequipotent at a 1 ng dose (FIG. 2). Hence, the biosynthesis of PD1 fromDHA, from the results of the present experiments, appears to requirestereoselective enzymatic steps to evoke bioactions. This requirementfor stereoselective enzymatic reactions is widely appreciated in thebiosynthesis of eicosanoids (9, 44). The nature of the PD1 epoxidehydrolase in vivo is therefore of interest, particularly in view of thebioactivity results with Compound VI, which was the most potent of theisomers (FIG. 6). Compound VI shares the triene geometry of PD1,differing only in the C10 chirality in the S configuration. However,only trace amounts of this isomer appear to be generated by human cells(vide infra). Thus the fidelity of the enzyme that produces PD1 from theproposed carbonium cation intermediate (1) in its ability to directinsertion of H₂O-derived alcohol at carbon 10 exclusively in the 10Rwith apparently trace amounts of 10S as in Compound VI (FIGS. 2 and 6)is an intriguing point for further studies.

Earlier results indicated that DHA, which is not a natural substrate forpotato 5-lipoxygenase, is converted to 10-HDHA by this enzyme and thedouble dioxygenation product 10,20-diHDHA (47). In addition, Whelan etal. demonstrated that this plant lipoxygenase is very versatile with DHAas a substrate (48) and identified multiple monohydroxy-DHA products atcarbon positions 4, 7, 8, 11, 13, 14, 16, 17 to give positional isomersof HDHA; each was an enzymatic product of this flexible enzyme. Thisregioselectivity also likely reflects the degree of enzyme purity aswell as the geographic source of the potato. It was found that potato5-LOX and soybean 15-lipoxygenase gave specific diHDHA profiles ofproducts that were dependent on pH, enzyme, and substrate concentrationsused in the incubations (3). When the substrates were presented inmicellar configuration with the enzymes, hydroperoxy intermediates wereconverted to epoxides that, on hydrolysis, gave many of the isomers asrelatively minor products but were nonetheless in quantities useful forin vitro and in vivo studies (37). These findings were advantageous inthe preparation of intermediates (i.e., 7,17-diHDHA, 17S—HDHA, and17S—H(p)DHA) used in biosynthesis studies and determining the identityand actions of enzymatic products generated in vivo as well as byisolated human cells from DHA (1, 3).

Recently, the chirality of the DHA potato 5-LOX product 10-HDHA,originally reported (47) by J. Whelan and C. Reddy in 1988, wasestablished as 10S—HDHA by classic steric analysis, and the formation of10,20-diHDHA and 17-H(p)DHA were reportedly optimized for the plantlipoxygenases (49). In the present studies, the double dioxygenationproduct prepared, matched, and identified in both suspensions of humanPMN (see FIG. 1A, m/z 261 profile) and in vivo during peritonitiscarries its alcohols as expected in the 10S,17S configuration in thisdiHDHA (Compound I). Hence, this natural isomer of PD1 (Compound II)formed in vivo from DHA has its Δ13 position double bond in the cisconfiguration (i.e., 13Z) and its Δ11 in the trans configuration withinthe conjugated triene portion of the molecule (11E,13Z,15E) andpossesses some anti-inflammatory activity in vivo, albeit proved to bemuch less potent than natural or synthetic PD1 (Compound II). The humanand murine enzymes(s) involved in the biosynthesis of 10S,17S-diHDHA,the dioxygenation product, have not been determined.

In peritonitis, PD1 significantly reduced PMN infiltration at doses aslow as 100 ng/mouse that reached an apparent maximal response at ˜50%range (1). This level of inhibition of PMN infiltration may be relatedto PD1's endogenous anti-inflammatory roles in physiologic settings andthus relevant in dampening PMN infiltration in inflammation as a naturalmechanism rather than complete inhibition of PMN transmigration, anevent that in theory could lead to immune suppression of microbial hostdefense mechanisms. In the present studies, it was confirmed that7,17-diH(p)DHA (1, 3) and 10,17-diH(p)DHA (49) are both doubledioxygenation products (see FIG. 4). ¹⁸O was incorporated at the carbon10-position alcohol that originated from enriched atmosphere molecular¹⁸O₂ via a lipoxygenase mechanism (FIG. 4A). The evidence for ¹⁸Oincorporation at carbon 10 position includes the 2 amu increase inprominent ions in the mass spectrum of 10,17-diHDHA, e.g., m/z 299, 263,183, 343 (cf FIGS. 1B and C) that was obtained with either 17S—H(p)DHAor 17S—HDHA as substrates. The 10-position alcohol results fromlipoxygenation and with native DHA as sequential actions oflipoxygenase(s) (FIG. 4B), since the chirality at the 10-position islikely in predominantly the S configuration (Compound I), the remainderin the 10R configuration as in Compound IV. (see ref. 1).

In peritonitis, PD1 significantly reduced PMN infiltration at doses aslow as 100 ng/mouse that reached an apparent maximal response at ˜50%range (1). This level of inhibition of PMN infiltration may be relatedto PD1's endogenous anti-inflammatory roles in physiologic settings andthus relevant in dampening PMN infiltration in inflammation as a naturalmechanism rather than complete inhibition of PMN transmigration, anevent that in theory could lead to immune suppression of microbial hostdefense mechanisms. In the present studies, it was confirmed that7,17-diH(p)DHA (1, 3) and 10,17-diH(p)DHA (49) are both doubledioxygenation products (see FIG. 4). ¹⁸O was incorporated at the carbon10-position alcohol that originated from enriched atmosphere molecular¹⁸O₂ via a lipoxygenase mechanism (FIG. 4A). The evidence for ¹⁸Oincorporation at carbon 10 position includes the 2 amu increase inprominent ions in the mass spectrum of 10,17-diHDHA, e.g., m/z 299, 263,183, 343 (cf FIGS. 1B and C) that was obtained with either 17S—H(p)DHAor 17S—HDHA as substrates. The 10-position alcohol results fromlipoxygenation and with native DHA as sequential actions oflipoxygenase(s) (FIG. 4B), since the chirality at the 10-position islikely in predominantly the S configuration (Compound I), the remainderin the 10R configuration as in Compound IV.

At the 1 ng/mouse dose, 10S,17S-diHDHA (Compound I) did display someactivity but this activity did not increase with higher doses in astatistically significant fashion. Also, this double dioxygenationisomer 10S,17S-diHDHA (Compound I) was not active at the 0.1 ng dosecompared to PD1. Given the double bond geometry determined in thepresent study for the conjugated triene unit of PD1 as Δ11E,13E,15Z and10-position alcohol in the R configuration (FIGS. 1 and 2 and FIG. 8),it is likely that, once the 16(17)-epoxide intermediate is produced from175-H(p)DHA (1), it is enzymatically subject to hydrolysis. This opensattack of the proposed cation intermediate by water-derived oxygenrather than molecular oxygen in vivo to give the 10R configuration andset the triene double bond configuration to trans, trans, cis geometryat Δ11E,13E,15Z in PD1. This enzymatic mechanism is also supported byidentification of epoxide-derived alcohol trapping products in humanleukocytes and glial cells and the isolation and identification of twovicinal diols as minor hydrolysis products, namely16,17-dihydroxydocosatrienes (see ref. 1). Further studies are warrantedto identify the enzyme(s) and establish their role in PD1 biosynthesisas noted above.

In earlier experiments, when administered i.v., 10,17S-docosatriene(PD1, Compound II, FIG. 2) was found to be more potent than indomethacinin reducing PMN infiltration in murine peritonitis, i.e., ˜40%inhibition at 100 ng/mouse (1). Synthetic PD1 in as small a dose as 1ng/mouse gave ˜40% inhibition of PMN infiltration that was maintained atthe ng and 100 ng doses. Thus, synthetic PD1 (Compound II) matched withthe natural compound is a potent regulator of PMN infiltration in vivobut does not completely block PMN recruitment, which is consistent withits counterregulatory and autacoid actions and apparently would notcompromise host defense via immune suppression of effector cellfunction. This was also the case with human PMN transmigration, whichrequired 10-100 nM PD1 to reduce PMN transmigration by 35-45% in vitro.Thus, although PD1 stereoselectively reduces PMN transmigration invitro, given its potent actions in vivo, PD1 (Compound II) likelytargets additional cell types in vivo to evoke its potentanti-inflammatory actions in vivo (FIG. 7 and FIG. 9). In murineperitonitis, PD1 also regulated both monocyte and lymphocyte traffic tothe exudates. Alternatively, these potencies in vivo vs. isolated humancells might also reflect species differences. As an inducer ofperitonitis, zymosan stimulates the initial formation of many endogenouschemoattractants for PMN, i.e., LTB₄, the complement component C5a,chemokines and cytokines (37). Since PD1 stops PMN recruitment in vivo,it counteracts these several different sets of PMN chemoattractants thatregulate trafficking of these cells in vivo (FIG. 9 and see ref. 37).Given the inherent chemical liabilities of PD1, a more chemically stableform denoted 15,16-dehydro-PD1 (FIG. 9) was prepared and tested.Although less potent, chemical stabilization of the conjugated doublebonds with an acetylenic form proved useful as the molecule retainedactivity in vivo (FIG. 9). These results are consistent with the ˜40%inhibition obtained with a 4,5-acetylenic analog of PD1 (37).

In addition to this lipoxygenase-initiated route of biosynthesis forPD1, an aspirin-triggered route with a 17R epimer of PD1 (17R series) isgenerated via acetylated COX-2 and subsequent reactions (3, 32); thecomplete stereochemistry of this bioactive epimer is in progress. It isof interest to note that compound V,10S,17R-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (FIG. 2)was essentially inactive in vivo (FIG. 6A). Whether the many beneficialactions reported for DHA in vitro and with DHA dietary supplementationin humans (21, 24-26) are linked to the formation and actions of thesenew families of DHA-derived mediators, protectin and D-series resolvins,is of interest and a timely proposal in view of the importance ofuncontrolled inflammation in many widely occurring human diseases. Thesefindings also underscore yet another similarity between the immune andneural systems. Hence, results of the present experiments establish thestereochemistry of PD1 (Compound II) and its natural isomers generatedby human leukocytes and murine tissues during inflammation. Moreover,they confirm the potent stereoselective anti-inflammatory actions of PD1and provide new avenues to mark the impact of DHAutilization/supplementation and its endogenousanti-inflammatory/proresolving actions by monitoring PD1, given itsunique physical and biological properties documented in the presentreport.

Evidence is provided herein for protectin D1(PD1,10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid)formation from docosahexaenoic acid in human asthma in vivo and PD1counter-regulatory actions in allergic airway inflammation. PD1 and17S-hydroxy-docosahexaenoic acid were present in exhaled breathcondensates from healthy subjects. Of interest, levels of PD1 weresignificantly lower in exhaled breath condensates from subjects withasthma exacerbations. PD1 was also present in extracts of murine lungsfrom both control animals and those sensitized and aerosol challengedwith allergen. When PD1 was administered prior to aeroallergenchallenge, airway eosinophil and T-lymphocyte recruitment weredecreased, as were airway mucus, levels of specific pro-inflammatorymediators, including interleukin-13, cysteinyl leukotrienes andprostaglandin D₂, and airway hyper-responsiveness to inhaledmethacholine. PD1 treatment after aeroallergen challenge markedlyaccelerated the resolution of airway inflammation. Together, thesefindings provide evidence for endogenous PD1 as a pivotalcounter-regulatory signal in allergic airway inflammation and point tonew therapeutic strategies for modulating inflammation in asthmaticlung.

Chronic airway inflammation with large numbers of eosinophils (EOS) andT lymphocytes (Lymphs) infiltrating respiratory tissues ismechanistically linked to asthma pathogenesis (50). In addition to theirdirect actions, these leukocytes amplify airway inflammation bytrafficking into the lung an increased capacity to generate bothpro-inflammatory peptides and lipid mediators, such as T_(H)2 cytokinesand cysteinyl leukotrienes (CysLTs) (50). In addition, T_(H)2 cytokinesup-regulate the expression of biosynthetic enzymes foreicosanoids—including prostaglandins (PGs), LTs and lipoxins (LXs) withpotent immunomodulatory properties (51).

DHA levels in the respiratory tract are decreased in asthma and otherdiseases of excess airway inflammation, such as cystic fibrosis (55).Epidemiologically, a diet high in marine fatty acids (fish oil) may havebeneficial effects on inflammatory conditions, including asthma (56, 57)and dietary supplementation with omega-3 fatty acids in childrenprevents the development of atopic cough, a symptom of allergic airwayinflammation (58). The underlying mechanisms for beneficial propertiesof omega-3 fatty acids in asthma remain to be established.

Natural resolution of acute inflammation (or asthma exacerbation) isdriven, in part, by decrements in pro-inflammatory mediators and removalof inflammatory cells (50, 59). Promotion of resolution is nowrecognized as an active process with early signaling pathways, forexample cyclooxygenase-2 derived PGE₂ and PGD₂, engaging biosyntheticcircuits for the later formation of counter-regulatory mediators, suchas LXs and the newly identified families of lipid mediators generatedfrom omega-3 fatty acids named resolvins and protectins that candominate the resolution phase (60). DHA is incorporated into membranesand rapidly released upon neuronal cell activation (61) for conversionto 17S-hydroxy containing resolvins of the D series (because they arefrom DHA) and protectin D1 (PD1) (62). The complete stereochemistry forPD1 is established as10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid.Throughout the specification, it is shown that PD1 is generated fromendogenous sources in human asthma and reduces both allergic airwayinflammation and hyper-responsiveness.

In one embodiment, the invention provides PD1 and analogs thereof havingthe formula:

wherein R is a hydrogen atom, an alkyl group, or is a pharmaceuticallyacceptable salt and each of P₁ and P₂, individually, is a hydrogen atomor a protecting group. In one particular aspect, when P₁ and P₂ are bothhydrogen atoms, then R is a pharmaceutically acceptable salt or an alkylgroup, such as a methyl or ethyl group. In another aspect, when P₁ andP₂ are both hydrogen atoms, then R can also be a hydrogen atom providedthat the compound is isolated or substantially purified.

In other aspects, the invention provides10,17-dihydroxy-docasa-hexaenoic acid derivatives having the generalformula (VII):

wherein R is a hydrogen atom, an alkyl group, or is a pharmaceuticallyacceptable salt and each of P₁ and P₂, individually, is a hydrogen atomor a protecting group. The dashed line represents that the double bondcan be “cis” or “trans” in configuration. In certain aspects, compoundsI, II, IV, V and VI are excluded from the invention.

In still another aspect, the invention provides10,17-dihydroxy-15,16-dehydrodocasahexaenoic acid derivatives having thegeneral formula (VIII):

wherein R, P₁ and P₂, and the dashed lines are as described above. Thechiral centers at 10 and 17 can be R/S, S/R or mixtures thereof. Incertain embodiments, the 4,5 bond is cis, the 7,8 bond is cis, the 11,12bond is trans, the 13,14 bond is trans, the 15,16 bond is acetylenic andthe 19,20 bond is cis.

In still yet another aspect, the invention provides a10,17-dihydroxy-15,16-dehydrodocasahexaenoic acid derivative having thegeneral formula (IX):

wherein R, P₁ and P₂ are as described above.

In one aspect of the invention, the compound(s) of the invention aresubstantially purified and isolated by techniques known in the art. Thepurity of the purified compounds is generally at least about 90%,preferably at least about 95%, and most preferably at least about 99% byweight (100% by weight).

In certain embodiments, the subject compounds are purified, e.g.,substantially separated from other compounds or isomers that are presentin a cellular environment where resolvins are produced or that arepresent in crude products of synthetic chemical manufacturing processes.In certain embodiments, a purified compound is contaminated with lessthan 25%, less than 15%, less than 10%, less than 5%, less than 2%, oreven less than 1% of cellular components (proteins, nucleic acids,carbohydrates, etc.), chemical byproducts, reagents, and startingmaterials, and the like. In certain embodiments, a purified compound iscontaminated with less than 25%, less than 15%, less than 10%, less than5%, less than 2%, or even less than 1% of other resolvins and/or otherisomers of the compound. The addition of pharmaceutical excipients,other active agents, or other pharmaceutically acceptable additives isnot understood to decrease the purity of a compound as this term is usedherein.

The compounds described throughout the specification can be administeredalone or in combination with a pharmaceutically acceptable carrier.

The compounds described throughout the specification can be used totreat inflammation, and in particular airway inflammatory conditionssuch as asthma.

The compounds described throughout the specification can be administeredalone or in combination with one another.

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C1-C6 means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl; cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. In preferredembodiments, the allyl groups are (C1-C6) alkyl.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like. Inpreferred embodiments, the alkanyl groups are (C1-C6) alkanyl.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic allyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). Typical alkenylgroups include, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like. In preferred embodiments, the alkenyl group is (C2-C6)alkenyl.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. In preferredembodiments, the alkynyl group is (C2-C6) alkynyl.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene and Wuts,Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, John Wiley &Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogenprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxyl protecting groupsinclude, but are not limited to, those where the hydroxyl group iseither acylated (esterified) or alkylated such as benzyl and tritylethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilylethers (e.g., TMS or TIPPS groups), glycol ethers, such as ethyleneglycol and propylene glycol derivatives and allyl ethers.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a compound(s) of thepresent invention within or to the subject such that it can perform itsintended function. Typically, such compounds are carried or transportedfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations.

In certain embodiments, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts, esters,amides, and prodrugs” as used herein refers to those carboxylate salts,amino acid addition salts, esters, amides, and prodrugs of the compoundsof the present invention which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of patientswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use of the compounds of the invention. The term “salts”refers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds orby separately reacting the purified compound in its free base form witha suitable organic or inorganic acid and isolating the salt thus formed.These may include cations based on the alkali and alkaline earth metals,such as sodium, lithium, potassium, calcium, magnesium and the like, aswell as non-toxic ammonium, quaternary ammonium, and amine cationsincluding, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. (See, for example, Berge S. M.,et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977; 66:1-19 which isincorporated herein by reference).

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. The term isfurther intended to include lower hydrocarbon groups capable of beingsolvated under physiological conditions, e.g., alkyl esters, methyl,ethyl and propyl esters.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred percent,this amount will range from about 1 percent to about ninety-nine percentof active ingredient, preferably from about 5 percent to about 70percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention. Suchsolutions are useful for the treatment of conjunctivitis.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Intravenous injection administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systematically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracistemally and topically, as by powders, ointments ordrops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of ordinary skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated analgesic effects, will range from about0.0001 to about 100 mg per kilogram of body weight per day, morepreferably from about 0.01 to about 50 mg per kg per day, and still morepreferably from about 0.1 to about 40 mg per kg per day. For example,between about 0.01 microgram and 20 micrograms, between about 20micrograms and 100 micrograms and between about 10 micrograms and 200micrograms of the compounds of the invention are administered per 20grams of subject weight.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The pharmaceutical compositions of the invention include a“therapeutically effective amount” or a “prophylactically effectiveamount” of one or more of the compounds of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, e.g., a diminishment or prevention of effectsassociated with various disease states or conditions. A therapeuticallyeffective amount of the compound may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the therapeutic compound to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the therapeutic agent are outweighed bythe therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a compound of the invention is0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted that dosagevalues may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

Delivery of the compound of the present invention to the lung by way ofinhalation is an important method of treating a variety of respiratoryconditions (airway inflammation) noted throughout the specification,including such common local conditions as bronchial asthma and chronicobstructive pulmonary disease. The compound can be administered to thelung in the form of an aerosol of particles of respirable size (lessthan about 10 μm in diameter). The aerosol formulation can be presentedas a liquid or a dry powder. In order to assure proper particle size ina liquid aerosol, as a suspension, particles can be prepared inrespirable size and then incorporated into the suspension formulationcontaining a propellant. Alternatively, formulations can be prepared insolution form in order to avoid the concern for proper particle size inthe formulation. Solution formulations should be dispensed in a mannerthat produces particles or droplets of respirable size.

Once prepared an aerosol formulation is filled into an aerosol canisterequipped with a metered dose valve. The formulation is dispensed via anactuator adapted to direct the dose from the valve to the subject.

Formulations of the invention can be prepared by combining (i) at leastone compound of the invention in an amount sufficient to provide aplurality of therapeutically effective doses; (ii) the water addition inan amount effective to stabilize each of the formulations; (iii) thepropellant in an amount sufficient to propel a plurality of doses froman aerosol canister; and (iv) any further optional components e.g.ethanol as a cosolvent; and dispersing the components. The componentscan be dispersed using a conventional mixer or homogenizer, by shaking,or by ultrasonic energy. Bulk formulation can be transferred to smallerindividual aerosol vials by using valve to valve transfer methods,pressure filling or by using conventional cold-fill methods. It is notrequired that a stabilizer used in a suspension aerosol formulation besoluble in the propellant. Those that are not sufficiently soluble canbe coated onto the drug particles in an appropriate amount and thecoated particles can then be incorporated in a formulation as describedabove.

Aerosol canisters equipped with conventional valves, preferably metereddose valves, can be used to deliver the formulations of the invention.Conventional neoprene and buna valve rubbers used in metered dose valvesfor delivering conventional CFC formulations can be used withformulations containing HFC-134a or HFC-227. Other suitable materialsinclude nitrile rubber such as DB-218 (American Gasket and Rubber,Schiller Park, Ill.) or an EPDM rubber such as Vistalon™ (Exxon),Royalene™ (UniRoyal), bunaEP (Bayer). Also suitable are diaphragmsfashioned by extrusion, injection molding or compression molding from athermoplastic elastomeric material such as FLEXOMER™ GERS1085 NTpolyolefin (Union Carbide).

Formulations of the invention can be contained in conventional aerosolcanisters, coated or uncoated, anodized or unanodized, e.g., those ofaluminum, glass, stainless steel, polyethylene terephthalate.

The formulation(s) of the invention can be delivered to the respiratorytract and/or lung by oral inhalation in order to effect bronchodilationor in order to treat a condition susceptible of treatment by inhalation,e.g., asthma, chronic obstructive pulmonary disease, etc. as describedthroughout the specification.

The formulations of the invention can also be delivered by nasalinhalation as known in the art in order to treat or prevent therespiratory conditions mentioned throughout the specification.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

The invention features an article of manufacture that contains packagingmaterial and a compound of the invention contained within the packagingmaterial. This formulation contains an at least one compound of theinvention and the packaging material contains a label or package insertindicating that the formulation can be administered to the subject totreat one or more conditions as described herein, in an amount, at afrequency, and for a duration effective to treat or prevent suchcondition(s). Such conditions are mentioned throughout the specificationand are incorporated herein by reference. Suitable EPA analogs and DHAanalogs are described herein.

More specifically, the invention features an article of manufacture thatcontains packaging material and at least one compound of the inventioncontained within the packaging material. The packaging material containsa label or package insert indicating that the formulation can beadministered to the subject to asthma in an amount, at a frequency, andfor a duration effective treat or prevent symptoms associated with suchdisease states or conditions discussed throughout this specification.

Materials and Methods

Materials—Zymosan A, soybean lipoxygenase (fraction V), and calciumionophore, A-23187, were purchased from Sigma Co. (St. Louis, Mo.).Docosahexaenoic acid (C22:6, DHA) and 5-LO from potato (pt5LO) were fromCayman Chemical Co. (Ann Arbor, Mich.). Additional materials used inLC-UV-MS-MS analyses were from vendors reported in (1, 3). ¹⁸O₂ isotopewas purchased from Cambridge Isotopes (Andover, Mass.).

Isolation, LC-MS-MS and GC-MS Analyses—Incubations were extracted withdeuterium-labeled internal standard (PGE₂) (Cayman Chemicals) forLC-MS-MS analysis using a Finnigan LCQ liquid chromatography ion traptandem mass spectrometer equipped with a LUNA C18-2 (150×2 mm 5 μm)column and a rapid spectra scanning UV diode array detector using mobilephase (methanol:water:acetate at 65:35:0.01) with a 0.2 ml/min flow ratethat monitored UV absorbance ˜0.1 min before samples entered the MS-MS.The scan acquisition rates were 11/min for MS-MS and 60/min for UV,which give rise to a lag interval in retention times that was correctedin the results presented for each molecule. All intact cell incubationsand in vivo exudates were stopped with 2 vol cold methanol and kept at20° C. for >30 min. Samples were extracted using C18 solid phaseextraction and further analyzed using gas chromatography-massspectrometry (GC-MS) using a Hewlett-Packard 6890 with a HP 5973 massdetector (see FIG. 8), and tandem liquid chromatography-massspectrometry (LC-MS-MS). Detailed procedures for isolation,quantitation, and structural determination of these DHA and relatedlipid-derived mediators were reported recently (1, 36) and used hereessentially as reported for elucidation of new products. Biogenicsynthesis of some of the DHA-derived products were performed usingisolated enzymes, i.e., 5-LOX from potato and 15-LO were each incubatedin tandem sequential reactions (see refs. 1, 3, 37, 38) with either DHA,17S-hydroxy-DHA, or 17S-hydroperoxy-DHA to produce the compounds inquantities suitable for isolation and incubation with cells and tissuesas well as confirmation of physical properties and assigning biologicalactions. Incubations in an ¹⁸O₂-enriched atmosphere were performed andanalyzed as in ref. (39).

NMR for Protectin D1—¹H NMR (400 MHz, MeOH-d4): δ 6.52 (dd, J=14.1 Hzand 11.8 Hz, 1H), 6.26 (m, 2H), 6.07 (dd, J=11.1 Hz and 11.1 Hz, 1H),5.50-5.28 (m, 7H), 4.90 (s, 2H), 5.50-5.60 (m, 2H), 4.55 (m, 1H), 4.14(m, 1H), 3.65 (s, 3H), 2.82 (m, 2H), 2.40-2.13 (m, 8H), 2.06 (m, 2H),2.07 (m, 2H), 0.96 (t, J=7.5 Hz, 3H).

¹³C NMR (125 Hz, MeOH-d4): δ 174.93, 137.59, 134.56, 134.47, 134.35,131.01, 130.52, 130.17, 129.92, 128.57, 128.52, 126.14, 124.89, 72.60,68.18, 36.00, 35.97, 34.45, 26.30, 23.43, 21.312, 14.20.

Incubations with Human PMN and Whole Murine Brain—Human venous wholeblood (˜10 ml) was collected into heparin (0.01 units/ml) viavenipuncture from healthy volunteers (who declined taking medication for˜2 weeks before donation; Brigham and Women's Hospital protocol88-02642). Human PMN were freshly isolated from whole blood using Ficollgradient and enumerated as in (40). PMN (30×10⁶ cells/ml) were exposedto ionophore A23187 (5 μM) with either DHA or 17S-hydro(peroxy)-DHA (15μg/ml). Cell suspensions were incubated for 30 min at 37° C. in acovered water bath. Lipidomics and lipid mediator profile analyses werecarried out for DHA-derived products, resolving, and docosatrienes as in(1, 3, 18, 36). Whole murine brains (Charles River Laboratories,Wilmington, Mass.) were excised, and then homogenized in PBS (minus Mg²⁺and Ca²⁺) (Cambrex Bioscience, Walkersville, Md.). Brain homogenateswere washed one time (800 rpm, 4° C., 5 min) in PBS, and then suspendedin 1 ml of PBS minus divalent cations. Homogenates were placed in theincubator (5 min) with an atmosphere of 5% CO₂, 37° C., and calciumionophore A23187 (5 μM) or DHA (30 μM) was added. Brain homogenates werethen placed in the incubator for 30 min with an atmosphere of 5% CO₂(37° C.). Incubations were stopped with 2 volumes of ice-cold MeOH, and,after 30 min at 4° C., the suspensions were pelleted and thesupernatants were extracted using solid phase C18 cartridges (AlltechAssociates, Deerfield, Ill.). Material eluted with methyl formate wastaken to dryness using a stream of nitrogen gas and taken for furtheranalyses.

Human Neutrophil Transmigration—PMN were freshly isolated from wholeblood obtained by venipuncture from healthy human donors (who deniedtaking medication for 2 weeks prior to donation; BWH protocol no.88-02642) and anti-coagulated with acid citrate dextrose as in (40, 41).Briefly, plasma and mononuclear cells were removed by aspiration fromthe buffy coat after centrifugation (400 g; 20 min) at room temperature.Histopaque (density 1.077) was from Sigma-Aldrich (St. Louis, Mo.). RBCswere sedimented using 2% gelatin, and residual RBCs were removed bylysis in ice-cold NH₄Cl buffer. The cell suspensions were >90% PMN asdetermined by light microscopic evaluation. PMN were suspended at 5×10⁷cells/ml in HBSS with 10 mM Hepes, pH 7.4, and without Ca²⁺ or Mg²⁺(Sigma-Aldrich, St. Louis, Mo.). PMN were used within 2 hours of theirisolation.

Human Microvascular Endothelial Cells (HMEC; a gift from FranciscoCandal of the Centers for Disease Control, Atlanta, Ga.) were obtainedas primary cultures. For preparation of experimental HMEC monolayers,confluent endothelial cells were grown on 0.33 cm² ring-supportedpolycarbonate filters (5 μm pore size; Costar Corp., Cambridge, Mass.)in the apical-to-basolateral direction. Cells were grown for ˜1 weekprior to transmigration experiments.

Transmigration was conducted essentially as in ref. (41). PMN wereincubated with either vehicle-containing buffer or compound for 15minutes at 37° C. A chemotactic gradient was established by placing HMECinserts that had been washed in HBSS with Ca²⁺ and Mg²⁺ (denoted +/+) in10⁻⁸M LTB₄ in the lower chambers. Neutrophils (10⁶ cells) were added to50 μl HBSS+/+ in the upper chambers and cells were incubated at 37° C.for 90 min. Transmigrated PMN were quantified by assessing the PMNazurophilic marker myeloperoxidase and a calibration curve. PMN werelysed by the addition of Triton X-100 to a final concentration of 0.5%.The samples were acidified with citrate buffer (final concentration 100mM, pH 4.2). An aliquot of each sample was added to an equal volume ofABTS solution (1 mM ABTS[2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)], 0.03% H₂O₂, 100mM sodium citrate buffer, pH 4.2) in a 96-well plate. The resultingcolor was monitored using a plate reader at 405 nm and a calibrationcurve for cell number.

Acute Inflamatory Exudates: Murine Peritonitis—Peritonitis was carriedout using 6-8 week-old FVB male mice (Charles River Laboratories) fedlaboratory Rodent Diet 5001 (Purina Mills) that were anesthetized withisoflurane, and compounds to be tested were administeredintraperitoneally. Zymosan A in 1 ml saline (1 mg/ml) was injected˜1-1.5 min later in the peritoneum. Each compound tested or vehiclealone was suspended in 1 μl ethanol and mixed in sterile saline (120μl). After intraperitoneal injections (either 2 or 4 hours of acuteinflammation), the mice were sacrificed in accordance with the HarvardMedical Area Standing Committee on Animals protocol no. 02570, andperitoneal lavages were collected rapidly and placed in an ice bath (4°C.) for enumeration, differential counts, and further analysis.

Sensitization and challenge protocols—Five to seven week old male FvBmice (Charles River Laboratories) were housed in isolation cages underviral antibody-free conditions. Mice were fed a standard diet(Laboratory Rodent Diet 5001, PMI Nutrition International) thatcontained no less than 4.5% total fat with 0.26% omega-3 fatty acids and<0.01% C20:4. After Harvard Medical Area IRB approval (Protocol #02570),mice were sensitized with intraperitoneal injections of ovalbumin (OVA)(Grade III; Sigma Chemical Co.) (10 μg) plus 1 mg aluminum hydroxide(ALUM) (J.T. Baker Chemical Co.) as adjuvant in 0.2 ml PBS on days 0 and7. On days 14, 15, 16 and 17, the mice received PD1 (2, 20 or 200 ng) (aproduct of biogenic synthesis (52)) or PBS with 1.6 mM CaCl₂ and 1.6 mMMgCl₂ (0.1 ml) by intravenous injection 30 min prior to an aerosolchallenge containing either PBS or 6% OVA for 25 min/day. Matchingexperiments were performed with 20 ng PD1 prepared by total organicsynthesis (62).

On day 18, 24 h after the last aerosol challenge, either airwayresponsiveness to aerosolized methacholine (0, 20, 50 and 75 mg, 10 sec)was measured, bilateral bronchoalveolar lavage (BAL) (2 aliquots of 1 mlPBS plus 0.6 mM EDTA) was performed or tissues were harvested forhistological analysis. Lung resistance was measured using a Flexiventventilator (SciReq). Resistance was measured as a function of time foreach animal, and peak and average values for each dose of methacholinewere recorded. No BAL or histological analysis was performed on thoseanimals undergoing airway hyper-responsiveness or lipid extractionstudies.

In select experiments, animals were sensitized and aeroallergenchallenged for four days prior to receiving PD1 (20 ng) or PBS byintravenous injection (0.1 ml) on days 18, 19 and 20. On day 21,bilateral BAL was performed.

Allergen-initiated respiratory inflammation—Murine lungs were fixed in10% buffered formalin and paraffin embedded for hematoxylin and eosinand periodic acid Schiff staining (Sigma Chemical Co.). Tissuemorphometry was performed by a member (K. Haley) of the LungHistopathology Core Laboratory at Brigham and Women's Hospital who wasblinded to the experimental conditions prior to histological analyses.Three fields per slide were examined at 200× magnification for vessels,large airways and alveoli with EOS counted at 400× magnification inrandomly assigned fields. Vessels were identified by perivascular smoothmuscle, and large airways were identified by at least ½ their diametereither cuboidal or columnar epithelia. Measurement of inflammatorymediators was determined in cell-free BAL fluid (BALF) (2000 g, 10 min)by sensitive and specific ELISAs, in tandem, for interleukin-5 (IL-5),IL-12, IL-13, PGD₂ (R&D Systems), cysteinyl LTs, (Cayman Chemical Co.),and LXA₄ (Neogen). Cells in BALF were resuspended in PBS, enumerated byhemocytometer, and concentrated onto microscope slides by cytocentrifuge(STATspin) (265 g). Cells were stained with a Wright-Giemsa stain (SigmaChemical Co.) to determine leukocyte differentials (after counting≧200cells).

PD1 Extraction and Identification by LC-MS-MS—Exhaled breath condensateswere collected by R-tube (Respiratory Research, Inc.) from volunteersubjects who had given written informed consent to a protocol approvedby the Brigham and Women's Hospital Committee for the Protection ofHuman Subjects in Research. Samples were collected during 10 minutes oftidal breathing. Characteristics of the subjects are provided in FIG.10. For samples of murine lung, blood was flushed from the pulmonarycirculation with 2 ml PBS, and whole murine lungs were removed fromOVA-sensitized/OVA-challenged and control mice on Day 18. Using a manualdounce, lungs were gently homogenized for direct lipid extraction inMeOH or in some cases were warmed (5 min, 37° C.) in PBS, and incubated(40 min, 37° C.) in the absence or presence of DHA (100 μg). Reactionswere stopped with 10 volumes of iced MeOH and stored at −20° C.overnight.

Lipids in EBC or murine lung were extracted using C18 cartridges(Alltech) and deuterium-labeled d₄-PGE₂ as an internal standard tocorrect for losses during extraction (52). Materials eluting in themethyl formate fraction were taken to HPLC coupled to aphoto-diode-array detector and tandem mass spectrometry (LC-PDA-MS-MS,ThermoFinnigan) for lipidomic analyses. PD1 was identified usingcriteria that include retention time, coelution with authentic10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid, UVabsorbance in methanol (λmax 270 nm with shoulders at 261 and 281 nm, atriple band of absorption consistent with a conjugated triene structure)and at least 5 diagnostic MS-MS ions (m/z 359 [M-H], 341 ([M-H]-H₂O;base peak), 323 ([M-H]-2H₂O), 315 ([M-H]-CO₂), 297 ([M-H]-H₂O, —CO₂),and 277 ([M-H]-H₂-2H₂O—CO₂) plus additional ions defining the presenceof the C10 and C17 hydroxyl (i.e., m/z 289, 261, 243 (261-H₂O), 217(261-CO₂), 205, 181, 163 (181-H₂O) and 153) (FIG. 11). The quantitationof PD1 was determined following LC-MS-MS analyses using a calibrationcurve (r²=0.991) and the chromatographic peak areas obtained viaselective ion monitoring.

Statistical analysis—Results are expressed as the mean±SEM. Statisticalsignificance of differences was assessed by Student's t-test andKruskal-Wallis nonparametric one-way ANOVA. P<0.05 was set as the levelof significance.

Results

Complete Stereochemistry of PD1—The DHA-derived 10,17-dihydroxyconjugated triene-containing product PD1 is generated by several humancell types, murine exudates, skin, and brain tissues (1-3, 32), as wellas isolated fish tissues, indicating that it is a conserved structure inevolution (35). PD1 displays potent protective and anti-inflammatoryactions (1, 2, 18, 32). To determine the complete stereochemicalassignment of PD1, i.e., the chirality at C10 and the geometry of thetriene, the physical and biologic properties of DHA-derived PD1 andrelated 10,17 dihydroxy-docosatriene stereoisomers were directlycompared to those prepared by total organic and biogenic synthesis.These included: I 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid; II10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic-acid and V10S,17R,-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic-acid coelutein this HPLC system; III10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15E,19Z-hexaenoic-acid; IV10R,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic-acid; and VI10S,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic-acid. The totalsynthesis of these will be reported elsewhere.³ Of interest, I and VIcoeluted in this system, as did both II and V (FIGS. 1 and 2). BioactivePD1 was generated by isolated human cells, murine brain tissue, andduring inflammation in vivo. FIG. 1 reports representativechromatographic profiles for PD1 generated by isolated human neutrophilsincubated with DHA that was separated into several positional andgeometric isomers (FIG. 1A, top panel). The main positional isomer of10,17S-docosatriene was 7,17-diHDHA (denoted resolvin D5), as documentedearlier (1, 3), and a double dioxygenation product (see Ref. 37). Also,a representative profile of products is given for those obtained frommurine inflammatory exudates (FIG. 1A, middle panel) and neural tissues(not shown). A direct comparison of these materials is reported in PanelA, FIG. 1 along with the profile obtained for synthetic materials (FIG.1A, lower panel) and chromatograms recorded by MS-MS (right) and UV at270 nm (FIG. 1A, left side).

The complete stereochemistry of bioactive 10,17-docosatriene, PD1,namely the double bond geometry of the conjugated triene unit andchirality of its carbon 10-position remained to be established (see FIG.2, top, middle). In order to assign the complete stereochemistry ofbioactive PD1 and its related natural geometric isomers, it wasnecessary to carry out total organic synthesis and side-by-side matchingexperiments with murine and human systems because PD1 is generated inonly nanogram quantities commensurate with its potent actions in vivoand in vitro, but preclude direct NMR analysis. The mixture of syntheticisomers used is shown in the lower insert of Panel A, FIG. 1. The humanand murine generated bioactive PD1 matched the physical and biologicproperties of synthetic10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (CompoundII). In addition to both LC-MS-MS and GC-MS analyses with thesematerials (FIG. 8), experiments were carried out with both biologic andsynthetic isomers prepared with the same overall backbone structure,namely 10,17-dihydroxydocosatriene. A 17R-containing isomer was preparedand included in these experiments (isomer V; see FIG. 2), but could beeliminated in these assignments, since 17R-containing products are themajor series produced from DHA when aspirin is used (3, 18). Thus,although II and V coelute in this system, V could be eliminated as amajor DHA-derived product in these incubations (FIGS. 1 and 2).

The chirality of the alcohols and double bond geometry of the trienewere systematically addressed. FIG. 1B shows the MS-MS spectrum of PD1obtained from murine peritonitis (4 h) generated in vivo upon challengewith zymosan A. FIG. 1, Panel C reports the mass spectrum recorded usingthe same instrument settings and conditions with synthetic10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (CompoundII, FIGS. 1 and 2). To obtain additional evidence for matching, GC-MSanalyses were performed. FIG. 1, Panel D reports a representative massspectrum and prominent ions obtained with GC-MS for PD1 treated withdiazomethane and subsequently converted to its correspondingtrimethylsilyl derivative. Hence, chromatographic behavior and prominentions in two mass spectrometry systems (LC-MS-MS and GC-MS), togetherwith biological activity (see FIGS. 5 and 6), permitted criteria forassignment of the physical properties of PD1 and related isomers. Sincethe parent and daughter ions were the same for each isomer, retentiontime in two chromatographic systems and bioactivity were needed forassigning the stereochemistry of the endogenous PD1 (vide infra).

Compounds synthesized for these matching experiments are given in FIG.2. PD1 isolated and identified earlier carries alcohol groups at carbon10 and 17 positions flanking the conjugated triene portion of thismolecule (1, 3). The stereochemistry of the carbon 17-position alcoholwas retained from the precursor predominantly in the S configurationwhen derived from the LOX product 17S—H(p)DHA precursor (1, 3),eliminating isomer V from the matching panel in FIGS. 1 and 2. Thedouble bond geometry and stereochemistry of the alcohol group atposition 10 were tentatively assigned based on biogenic evidence, i.e.,the formation of alcohol trapping products in murine brain and humanleukocytes as well as identification of two vicinal diols 16,17S-diHDHA;hence the complete stereochemical assignment remained as illustrated inFIG. 2, top. To this end, each of the double bond isomers likely to bebiosynthesized was prepared in view of potential biosynthetic routesinvolved in PD1 formation, namely the involvement of epoxide-containingintermediates and/or double dioxygenation intermediates (1-3). The R andS configuration of the alcohol group at the carbon 10-position were eachprepared by stereocontrolled total organic synthesis. The strategy forthe synthesis of these is outlined in FIG. 3. Each of thestereocontrolled steps from defined precursors enabled preparation ofgeometric isomers of the conjugated triene region that were confirmed byNMR (see Materials and Methods). Also, for these experimentsdihydroxydocosanoids were prepared using isolated plant lipoxygenase(s)to obtain, as in earlier experiments (37), both positional isomers7,17S-diHDHA and 10,17S-diHDHA (1, 3). The preparation of these usingmicellar substrate was given in further detail in (37). These referencecompounds were useful in analyses of biosynthetic routes (see below).

FIG. 8 reports the prominent ions and chromatographic behaviors for eachof the double bond and positional isomers prepared (FIG. 2). Asexpected, each of these isomers gave characteristic UV λ_(max) ^(MeOH)for a conjugated triene chromophore with a λ_(max) ^(MeOH) at ˜270 nmwith shoulders at 260 nm and 282 nm (±2 nm). Each isomer gave a specificλ_(max) ^(MeOH), which appeared to reflect the geometry of the doublebond system. For example, the Δ15-trans isomer in the conjugated trieneportion of 10,17-diHDHA gave a UV λ_(max) ^(MeOH) of 269 nm (FIG. 8).Only one of these products (Compound II) matched the chromatographicbehavior using both LC-MS and GC-MS as well as biological activity. Asexpected, each of the major prominent ions for these isomers in bothLC-MS-MS and GC-MS were essentially identical (i.e., daughter and parentions were essentially the same for each).

The main materials isolated from human PMN coeluted with compound IIand, in some preparations, a trace amount of compound IV. Compound IVdiffers from II in its triene configuration, which is 11E,13Z,15E. Thischange in two double bonds was unlikely in view of the earlieridentification of alcohol trapping products (1). Also, compound IV wasnot observed in all PMN incubations, which might reflect some degree ofdonor variation. A second representative profile from another humandonor is reported in FIG. 1A, top right panel, at m/z 260.8 to 261.8 ofthe MS-MS for M-1 at m/z=359. As noted for this ion plot and donor,compound II is the most abundant and IV is not present. Also, compound Iis clearly present along with an unknown material denoted with anasterisk. In this panel, the retention time of isomer III is plotted ingray for direct comparison. Only trace amounts of III, the Δ15-transisomer of PD1, were routinely identified. The appearance of thisΔ15-trans isomer with its triene in the all-trans configuration likelyreflects workup-induced isomerization at the Δ15 position, which mayaccount for its varied presence in LC-MS-MS-based analysis. This is inaddition to Δ15-trans-PD1 formation via nonenzymatic hydrolysis of theproposed epoxide intermediate (FIG. 4B).

Compound I was consistently identified in profiles obtained from murineperitonitis (FIG. 1A middle). However, it was not the major product ofhuman cells nor did it carry potent actions as compound II did (FIGS. 5,6), also reported earlier (1, 3, 39). Consistent with its biosynthesis(vide infra), the appearance of this double dioxygenation product wastime-dependent in vivo and in vitro (not shown). Although isomer VIcoeluted with I in this system, it was excluded on the basis that it wasnot a major product of human cells and is not likely to be generatedfrom an epoxide intermediate without a specialized enzyme (see FIG. 4and below). The stereoselective insertion of oxygen from H₂O can beexpected to give rise to predominantly a 10R configuration whenattaching a carbonium cation intermediate; proposed in ref. (1).Compound VI differs from PD1 in its C10 position, which is 10S ratherthan 10R and is not a double dioxygenation product because its doublebond geometry in the triene portion of the compound is not consistentwith the biosynthesis of the triene in the trans, cis, transconfiguration. Since these and other lipid mediators are highlyconserved structures found in many species from fish to human (35), aspecies difference between mouse and human in PD1 structure is notlikely. Hence, although compound VI carries bioactivity (FIG. 6), it wasexcluded on the basis of the above findings and because compound VI wasnot a major isomer in human profiles, as compound II was. Hence,compound II matched PD1 formation and physical properties as well aspotency of action (see below).

The biosynthesis of 7,17-diHDHA in inflammatory exudates (3, 18) and itsformation from DHA or 17-hydroxy-DHA with isolated human neutrophilssuggested that the biosynthesis of this compound involved formation viadouble dioxygenation (18). That is, in addition to using molecularoxygen for insertion at the 17-position, lipoxygenation could alsoinsert molecular oxygen at the 7-position in sequential fashion. Theidentification of this novel compound from DHA and the sequentiallipoxygenation events in its formation (1, 3) appeared to be similar tothat of 5S,15S-diHETE generated from arachidonic acid (42, 43). Hence,it was of particular interest in earlier studies (37) when sequentialactions of potato 5-lipoxygenase and/or 15-lipoxygenase with thesubstrates in micellar configuration were noted to produce both7,17-diHDHA and 10,17-diHDHA isomers as major products as well asmultiple geometric isomers as minor products following hydrolysis ofenzymatically generated epoxides in vitro (cf. 1, cf. 3). The formationof the minor isomers was dependent on substrate, pH, and enzymeconcentration.

To test the role of sequential LO actions in the proposed mechanism ofPD1 formation (Compound II; see FIG. 2) and its isomer 10S,17S-diHDHA(Compound I), incubations were carried out in an atmosphere enriched inisotope ¹⁸O₂ with 17-hydroperoxy-DHA as substrate and isolated pt5-lipoxygenase (see Methods). Note that Compounds I and II differ inboth chirality at carbon 10 and geometry of their respective trieneconfigurations (FIG. 2). Following extraction and isolation, the productprofiles, GC-MS and LC-MS-MS results indicated that ¹⁸O was incorporatedin the carbon 10-position in 10S,17S-diHDHA (FIG. 4). Chromatographicseparation of 10S,17S-diHDHA (FIG. 4) gave prominent ions with MS-MS(FIG. 4, Panel A), indicating on average >75% incorporation of ¹⁸Ooriginating from molecular oxygen in the carbon 10-position with a rangeof 51.4 to 91.8% increase in diagnostic ions. Since these enzymes usemolecular oxygen as a substrate, it is not possible, under theseconditions, to completely replace enzyme-associated ¹⁶O for the ¹⁸Oisotope as calculated earlier for lipoxin A₄ in refs. (39, 44). Theextent of ¹⁸O present in diagnostic ions was determined for m/z 181/183,261/263, 289/291, 297/299, 315/317, 323/325, 341/343, and 359/361, andthe ratio of ¹⁶O to ¹⁸O calculated from ion intensities and averaged.These results indicate that 10S,17S-diHDHA can be produced via doublelipoxygenation.

Results for matching studies indicated that the double bond geometry forthe conjugated triene portion of this molecule was in the trans, cis,trans configuration (matching Compound I, FIG. 2). Hence, doubledioxygenation to form 10S,17S-diHDHA was also a mechanism to generatethis compound in vivo, since it is a prominent product in murineexudates from peritonitis, and to some extent present in suspensions ofhuman leukocytes incubated with DHA, (FIG. 1 and ref. 1) and murinebrain (3, 18), as well as trout leukocytes and brain (35). FIG. 4, PanelB outlines the proposed scheme and proposed role for doubledioxygenation and its products 10S,17S-diHDHA and 7S,17S-diHDHA. Thedouble bond geometry in the conjugated triene portion of the molecule(trans,cis,trans) is consistent with oxygenation using molecular oxygenwith two sequential lipoxygenation steps. Given the biological actions,chromatographic and physical properties of PD1 as well as the resultsfrom epoxide trapping experiments with human PMN and the isolation oftwo vicinal diol 16,17S-dihydroxy-docosatrienes as minor products (1),it is likely that, once a 16,17-epoxide-containing intermediate isgenerated in situ (as illustrated in FIG. 4), an enzymatic reaction isneeded to efficiently produce PD1 carrying the 10R,17S-dihydroxy-trans,trans, cis configuration arising from an epoxide intermediate asdepicted in FIG. 4, Panel B.

Anti-inflammatory Actions of PD1—As indicated above, the completestereochemical assignment for synthetic PD1 also relied on determiningbiological action of the related isomers. Earlier results indicated thatPD1's anti-inflammatory properties were comprised of blocking leukocyteinfiltration in murine systems (1, 3, 32, 37). Results in FIG. 5 showthat synthetic PD1 reduced PMN transmigration in response to leukotrieneB₄. Amounts as small as 1.0 μM gave 30% inhibition. The Δ15-trans isomerof PD1, where the conjugated triene portion of the molecule was in thetrans configuration, did not block PMN transmigration in vitro. AlthoughPD1 is a potent inhibitor in neutrophil transmigration, the degree ofinhibition observed with monolayers of human microvascular endothelialcells and human neutrophils from >5 separate donors did not achievevalues greater than 50% inhibition in each experiment.

These experiments with transmigration were carried out in parallel withmurine acute inflammation. In these, acute peritonitis was initiated bychallenge with the microbial isolate zymosan A and the actions of fiveisomers were assessed in vivo. Two compounds (Compound V and CompoundVI) were excluded from matching with PD1 because the physical retentiontimes on LC and GC-MS (FIG. 1 and FIG. 8) and biosyntheticconsiderations indicated that they were not likely candidates forendogenous human PD1 or isomers produced. It is noteworthy that PD1(Compound II, FIG. 2) at doses as low as 1 ng per mouse gave strikinginhibition of PMN infiltration within the exudates. In theseexperiments, the double dioxygenation product 10S,17S-docosatriene(Compound I) was substantially less potent. In this context, the doubledioxygenation product was not active at 0.1 ng compared directly tosynthetic PD1. At higher doses, 10S,17S—HDHA (Compound I) blocked PMNinfiltration, but it was less potent than PD1. Compound IV, which is the10R version of the double dioxygenation products, was essentiallyequipotent at a 1 ng dose (Compound IV≈Compound I) but did not increasepotency in a dose-dependent fashion at 10 ng and 100 ng doses (notshown). The Δ15-trans isomer of PD1 was, at equal doses of 1 ng/mouse,substantially less potent. Also, a rogue isomer for this series,Compound V (FIG. 2) was not likely to be produced in vivo from the17S-hydroxy precursor because its 10S,17R-diHDHA was essentially withoutactivity in this dose range. Of interest, Compound VI was the mostpotent of these isomers in vivo. However, only trace amounts were notedin human PMN extracts. Hence, a rank order of potency at the 0.1 ng doseof these 10,17-diHDHA isomers was Compound VI>>PD1>10S,17S-DT (thedouble dioxygenation product)>the Δ15-trans-PD1>>Compound V. The carboxymethyl ester of PD1 was also tested versus the native synthetic PD1.FIG. 6, Panel B demonstrates the potent dose response of PD1 as itdramatically reduced the infiltration of PMN into the peritoneum. Thecarbon 1-position carboxy-methyl ester was similar in its ability toblock in vivo the hallmark of acute inflammation, namely PMNinfiltration. The methyl ester of Compound VI also proved to be a potentregulator of PMN infiltration.

Can PD1 Stop Inflammation After Its Initiation?—Next, PD1 or its methylester was tested to determine if it could reduce leukocyte infiltrationonce inflammation had already been initiated. Results in FIG. 7Aindicate that doses as low as 1 ng PD1/mouse diminished infiltration ofPMN when administered i.p. following 2 h after challenge with zymosan invivo. Similar and striking results were obtained with the carboxy methylester of PD1, also administered i.p. Hence, once PD1 was given,essentially no further infiltration of PMN into the peritoneum wasobtained with essentially >90% blocking of further PMN infiltration tothe site. The anti-inflammatory actions of DHA-derived PD1 andEPA-derived resolvin E1 were evaluated for synergistic or additiveeffects in vivo. RvE1 is derived from EPA and is another omega-3-derivedcounterregulatory anti-inflammatory lipid mediator recently isolated andidentified (3, 45). When administered together, RvE1 and PD1 bothreduced the infiltration of PMN in vivo during zymosan-inducedperitonitis (FIG. 7B). These results indicate that they have a potentialadditive rather than synergistic anti-inflammatory action whenadministered together in vivo. A chemically more stable form ofsynthetic PD1, i.e., 15,16-dehydro-PD1, was prepared and tested thatproved to retain activity in vivo, reducing PMN infiltration, albeit wasslightly less potent than the native PD1 (FIG. 9). Of interest,differential counts on light microscopy also revealed that both PD1 andits chemical analog 15,16-dehydro-PD1 reduced PMN infiltration andincreased the non-phlogistic recruitment of monocytes and lymphocytes(FIG. 8) while reducing inflammation, a hallmark of resolution (17, 31).

PD1 is present in asthma and endogenously generated in allergic lung—Todetermine if DHA-derived products are generated in respiratory tissues,lipid extracts from exhaled breath condensates (EBCs) were analyzed thatwere collected from healthy volunteer subjects and patients in theemergency department during a clinical exacerbation of asthma (FIG. 10).PD1 and its biosynthetic precursor, 17(S)-hydroxy-DHA were present inthese human respiratory tract secretions (FIG. 11). Levels of PD1 weresignificantly lower in EBCs from subjects with status asthmaticus (traceamounts) compared to healthy individuals (2.23+/−1.55 ng PD1/ml EBC,mean+/−SEM, P<0.05). These results indicate that asthma exacerbation isassociated with reduced airway levels of the counter-regulatory lipidmediator PD1.

To investigate potential roles for PD1 in airway inflammation, anexperimental animal model of allergic asthma was studied. Aftersensitization and aerosol challenge with allergen, murine lungsgenerated PD1 from endogenous sources (73.9+/−35.6 ng PD1, mean+/−SEMfor n=3). Of note, PD1 levels in the inflamed lungs were notsignificantly different from those in healthy murine lungs (45.8 ngPD1). Similar to results with human EBCs, 17(S)-hydroxy-DHA was alsopresent in murine lungs. Addition of exogenous DHA to a homogenate ofthe inflamed murine lungs ex vivo significantly increased mean PD1levels by 5.8-fold to 431.6+/−69.3 ng PD1 (mean+/−SEM, n1=3, P<0.02).These findings indicate that during airway inflammation, respiratorytissues can convert DHA to 17(S)-hydroxy-DHA and PD1.

Allergic airway inflammation decreases with PD1—To determine the impactof PD1 on airway inflammation, physiologically relevant quantities (2,20 or 200 ng) were administered by intravenous injection toallergen-sensitized animals just prior (30 min) to each aerosolchallenge. For these experiments, PD1 was produced via biogenicsynthesis and matching studies were performed with PD1 that was preparedby total organic synthesis (62). Animals receiving PD1 had substantiallyless EOS and Lymphs in the peribronchial regions and airspaces comparedto control mice that received only vehicle (FIG. 12). PD1 also reducedgoblet cell hyperplasia and airway mucus as determined by periodic acidSchiff stain (FIG. 13). Morphometric analyses identified significantdecreases in EOS tissue infiltration around vessels and in the large andperipheral airways (FIG. 14 a). In BALF, PD1 decreased total leukocytes,EOS, and Lymphs in a concentration-dependent manner (FIG. 14 b), andlevels of peptide and lipid pro-inflammatory mediators were selectivelyreduced (FIG. 15). PD1 administration blocked allergen-induced increasesin IL-13, CysLTs and PGD₂, all of which have been assigned pivotal rolesin asthma pathobiology (63-65). Of note, PD1 did not significantlyimpact IL-5 or IL-12 levels in BALF. In conjunction with decreasedairway inflammation, levels of the counter-regulatory eicosanoid LXA₄were diminished in the presence of PD1 (FIG. 14 b). No behavioral orphysical signs of toxicity with PD1 treatment were observed. Together,these results indicate that PD1, in nanogram quantities, significantlyreduced allergic pulmonary inflammation, and suggests that its mechanismof action is distinct from LXs.

PD1 blocks airway hyper-responsiveness—Because increased airwayreactivity is a diagnostic hallmark of asthma, it was also determinedwhether PD1 regulated airway hyper-responsiveness to inhaledmethacholine. After allergen sensitization and aerosol challenge in thepresence of PD1 (0-200 ng), animals were ventilated and exposed (10 sec)to increasing concentrations of inhaled methacholine. Consistent withthe regulation of airway inflammation, PD1 also decreased both peak andaverage lung resistance in response to methacholine (FIG. 16). The logED₂₀₀ for the mean airway resistance for all three doses of PD1 (2, 20and 200 ng) was significantly increased compared with vehicle (FIG. 16b). There was a bell-shaped dose response with maximal protection forPD1 on airway hyper-responsiveness to methacholine apparent with loweramounts (2 and 20 ng). PD1 displayed no significant impact on the airwayresponses of control animals that received PBS rather than allergen(FIG. 16 b). In addition, no significant changes in lung elastance orcompliance were observed with PD1 following allergen challenge (data notshown). These results indicate that methacholine-inducedbronchoconstriction is significantly reduced by administration of PD1.

Impact of PD1 treatment on airway inflammation—To more closely mimic theclinical scenario of asthma exacerbation, it was next determined whetherPD1 could dampen established airway inflammation by administration afteraeroallergen challenge. Mice were sensitized and allergen challenged onfour consecutive days. PD1 (20 ng, iv) or vehicle (0.9% saline) was thengiven once a day for three additional days and BAL was performed toenumerate cellular infiltration into the lung. Despite no furtheraeroallergen challenges, animals receiving vehicle still carry asubstantial number of EOS and Lymphs in BALF at day 21 in our protocol(FIG. 17). In sharp contrast, PD1 administration led to significantdecrements in the numbers of total leukocytes, Eos and Lymphs in BALFs(FIG. 17). These findings indicated that PD1 has the capacity toaccelerate resolution of allergic airway inflammation.

PD1 is identified as a natural product of a new C22:6 signaling pathwayduring respiratory tract inflammation that displays potentcounter-regulatory actions on key asthma phenotypes, namely airwaylevels of pro-inflammatory peptide and lipid mediators, airway mucus,leukocyte accumulation and hyper-responsiveness. The present inventionprovides for the first identification of 17S-hydroxy-DHA and PD1 inhuman asthma. In addition, airway inflammation triggered PD1 formationin vivo and conversion of C22:6 to PD1 in lung tissues. Similar to theinflamed airway, generation of PD1 occurs elsewhere during multicellularhost inflammatory responses, including Alzheimer disease, brainischemia-reperfusion injury and activated human whole blood (52, 53,66). The biosynthesis of PD1 proceeds via 15-lipoxygenase-catalyzedconversion of DHA to 17S-hydroperoxy and 16(17)-epoxide intermediates inactivated human leukocytes and in Alzheimer's brain and murine cornea(53, 62, 67, 68). Lipoxygenases and epoxide hydrolases are bothprominent classes of enzymes in asthmatic lung that are induced bypivotal regulators of allergy, including specific T_(H)2 cytokines(69-72). Potential source(s) for PD1 generation include airwayepithelial cells, EOS and other leukocytes, but the definitive cellularand enzymatic source of PD1 in the lung remains to be established infuture studies. The present invention provides that the presence ofspecialized enzyme systems in the lung for this new DHA pathway thatconvert the omega-3 fatty acid to biologically active chemical mediatorsduring airway inflammation.

Eosinophilic airway inflammation and airway hyper-responsiveness arecharacteristic features of asthma. EOS recruitment to the lung in asthmais primarily a consequence of T_(H)2 lymph activation (50). Because PD1was identified in EBCs in the ng range and this sampling techniquelikely reflects only a small fraction of total PD1 generated in thelung, the impact of PD1 was examined in physiologically relevant ngquantities. After allergen sensitization and aerosol challenge, EOStrafficking was reduced by as little as 2 ng of PD1. Levels of T_(H)2cytokines in BALF and the number of Lymphs in both BALF and lung tissuewere decreased. These findings provide evidence for potent,concentration-dependent reduction of both T_(H)2 Lymphs and EOSresponses in vivo. Lymph and EOS activation in the lung are held tocontribute to asthma pathobiology. In addition, neutrophil (PMN)activation contributes to the pathogenesis of asthma exacerbation (73)and severity (74). PD1 promotes T-lymph apoptosis in vitro (67), and PD1also carries systemic and topical anti-inflammatory actions for PMNs invivo (52, 66). In the nervous system, PD1 decreases brain leukocyteinfiltration, IL-1β-induced NFκB activation and COX-2 expression toelicit neuroprotection (61, 66). Here, PD1 also dampenedhyper-responsiveness to methacholine and mucus production in theinflamed airway. The local generation of PD1 in allergic inflammationtogether with counter-regulatory properties in the airway broadens itspotential cellular sources and actions in vivo to new leukocyte classesand tissue resident cells and points to a more generalizedcounter-regulatory function as an autacoid in inflammation.

LXs are also generated in asthma and serve as potent inhibitors of bothairway inflammation and hyper-responsiveness (75). While there is someoverlap in the pattern of cytokine regulation for PD1 and a LX stableanalog in this murine model of asthma, some key differences wereobserved. First, while both mediators blocked IL-13 and CysLT generationand had no significant effect on BAL IL-12 levels (75), the inhibitoryconcentrations of PD1 were 1 to 2 log orders more potent than the LXanalog. Secondly, IL-5 production was reduced by the LX stable analog,but not PD1, suggesting a direct effect of PD1 on EOS, T Lymphs andother effector cells. Third, administration of PD1 led to decreasedairway levels of LXA₄, suggesting that the circuit for PD1 formation andactions is distinct from LX signaling in the murine lung. In aggregate,these findings indicate the presence of unique homeostatic pathways forDHA derived bioactive mediators in the lung.

It is interesting to note that formation of counter-regulatory LXs isdefective in severe inflammatory diseases of the airways, includingasthma and cystic fibrosis (76-78). DHA levels in the respiratory tractare decreased in both of these illnesses (55), and here, in comparisonto healthy controls, it was uncovered that there are lower levels of PD1during human asthma exacerbation. Given its counter-regulatoryproperties, decreased formation of PD1 from low levels of DHA wouldadversely impact control of airway inflammation andhyper-responsiveness. While observational studies have identified anincreased risk of asthma in populations with diets low in DHA,interventional trials with DHA supplementation have not consistentlyimproved clinical outcomes (79), despite altering the responses ofisolated leukocytes to inflammatory stimuli (80). In contrast,nutritional supplementation with omega-3 essential fatty acids hasproven beneficial in cystic fibrosis and the acute respiratory distresssyndrome, clinical disorders of excess PMN-mediated inflammation (81,82). Because the molecular rationale for these beneficial effects isuncertain, there remain many potential reasons for the lack of clinicalsuccess with DHA feeding in asthma, including purity, dose, time courseand difficulties tolerating the ingestion of large amounts of fish oilsfor extended periods of time (83). After the induction of experimentalasthma by aeroallergen challenge, we determined that administration ofPD1 promoted the resolution of airway inflammation. Thus, identificationof PD1 as a DHA-derived counter-regulatory autacoid in the lung opensthe door to new mechanism-based therapeutic strategies in airwayinflammation.

The present results are the first demonstration of PD1 formation inhuman asthma in vivo from DHA and identify direct protective andregulatory roles for this novel mediator in allergic inflammation andairway hyper-responsiveness. In light of its ability to strongly reduceboth of these key asthma phenotypes, the PD1 pathway may offer newtherapeutic approaches for asthma. Moreover, the results indicate thatendogenous conversion of DHA to PD1 represents a potential mechanism forthe therapeutic benefits derived from diets rich in this omega-3essential fatty acid in maintaining respiratory homeostasis.

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Although the present invention has been described with reference topreferred embodiments, persons skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention. All references cited throughout thespecification, including those in the background, are incorporatedherein in their entirety. Those skilled in the art will recognize, or beable to ascertain, using no more than routine experimentation, manyequivalents to specific embodiments of the invention describedspecifically herein. Such equivalents are intended to be encompassed inthe scope of the following claim.

1. A compound of formula (II):

wherein R is a hydrogen atom, an alkyl group, or a pharmaceuticallyacceptable salt; and P₁ and P₂ are each, individually, a hydrogen atomor a protecting group, provided when P₁ and P₂ are both hydrogen atoms,then R is other than a hydrogen atom.
 2. The compound of claim 1,wherein the compound is purified. 3-4. (canceled)
 5. A compositioncomprising a compound of claim 1 and a pharmaceutically acceptablecarrier.
 6. The composition of claim 5, wherein the compound ispurified. 7-8. (canceled)
 9. A method to treat airway inflammationcomprising administration of a therapeutically acceptable amount of acompound of claim 1
 10. (canceled)
 11. The method of claim 9, whereinsaid airway inflammation is asthma. 12-16. (canceled)
 17. A compound offormula (IX):

wherein R is a hydrogen atom, an alkyl group, or a pharmaceuticallyacceptable salt; and P₁ and P₂ are each, individually, a hydrogen atomor a protecting group.
 18. The compound of claim 17, wherein when P₁ andP₂ are both hydrogen atoms, then R is other than a hydrogen atom. 19.(canceled)
 20. The compound of claim 17, wherein P₁ and P₂ are eachhydrogen atoms and R is a hydrogen atom.
 21. A composition comprising acompound of claim 17 and a pharmaceutically acceptable carrier. 22-28.(canceled)
 29. A method to treat airway inflammation comprisingadministration of a therapeutically acceptable amount of a compound ofclaim
 17. 30. The method of claim 29, wherein P₁ and P₂ are eachhydrogen atoms and R is a hydrogen atom.
 31. The method of claim 29,wherein said airway inflammation is asthma.
 32. The method of claim 29,wherein when P₁ and P₂ are both hydrogen atoms, then R is other than ahydrogen atom.
 33. A method to treat airway inflammation comprisingadministration of a therapeutically acceptable amount of a compositionof claim
 21. 34. The method of claim 33, wherein P₁ and P₂ are eachhydrogen atoms and R is a hydrogen atom.
 35. The method of claim 33,wherein said airway inflammation is asthma.
 36. A method to treat airwayinflammation comprising administration of a therapeutically acceptableamount of a composition of claim
 5. 37. The method of claim 33, whereinP₁ and P₂ are each hydrogen atoms and R is a hydrogen atom.
 38. Themethod of claim 33, wherein said airway inflammation is asthma.